Amino acid residues in the second transmembrane (TM2) domain of nicotinic acetylcholine receptors (nAChRs) have been shown to have important regulatory functions in studies using site-directed mutagenesis and heterologous expression systems. This is true for sites in the beta subunit TM2 domain of both muscle and high-affinity neuronal nAChRs, as well as homopentameric alpha7 receptors. This proposal outlines a course of experimental study that will attempt to identify the possible roles of homologous sites in the alpha7 TM2 domain that have previously been established in the beta subunit as critical determinants of receptor function and pharmacology. This involves the substitution of amino acids from the muscle and neuronal beta subunit TM2 domains for identical residues in the alpha7 TM2 domain. Pharmacological characterization will include sensitivity to activation by agonist, sensitivity to non-competitive inhibitors (NCIs), and the degree of residual inhibition or desensitization. Functional characterization will involve analysis of receptor kinetics and divalent ion permeability. Thus far, the preliminary findings indicate that mutation of the alpha7 TM2 6' position to include either the muscle Beta1 subunit sequence or the neuronal Beta2/Beta4 subunit sequence, dramatically changes the response kinetics of the mutant receptors so that the decay rate of macroscopic currents is more like that of the respective wild-type beta subunit containing receptor than wild-type alpha7. The research plan outlined here will serve to further the training of the applicant in molecular biology and electrophysiology, and will help to identify the potential contribution of specific sites within the alpha7 TM2 domain to important aspects of its function and pharmacology.